We propose to develop a prognostic mRNA immune signature for resected stage II-III melanoma, in order to stratify these patients, who have a recurrence risk of ~50%. There is an urgent need to define accurate prognostic markers for stage II-III melanoma patients because adjuvant immunotherapy to prevent recurrence is both toxic and expensive. Unfortunately, conventional staging does not allow for accurate assessment of risk and many ?low risk? patients in fact progress. Further, while the immune tumor micro- environment is a key determinant of outcomes, standard pathologic assessment of tumor infiltrating lymphocytes (TILs) is subjective and not applicable to general clinical practice. In order to better stratify patients for adjuvant immunotherapy, we seek to validate a previously defined 53-gene signature. This signature employs NanoString, a probe based technology well suited to the analysis of the partially degraded RNA typically recovered from clinical grade FFPE tissue sections. We initially defined this signature in a training set and then validated these findings in an independent test set [Sivendran, et al. (2014) J. Invest. Dermatol. 134:2202-11]. As both training and test sets populations are retrospective, the next step to develop a clinically applicable assay is to test the signature on prospectively gathered samples. Given the fact that melanomas can recur years after resection in these early stage patients, prospective validation would be lengthy, and thus we opted to use the prospective retrospective analysis [PRA] approach whereby samples are collected and annotated prospectively but analyzed retrospectively. For this purpose we use samples from the Eastern Cooperative Group (ECOG) E1697 study of adjuvant interferon randomized vs placebo in stage II-III resected melanoma. Patients in this study have been maintained on study follow-up since they were randomized between 1998 and 2010. This project is collaboration between the Herbert Irving Comprehensive Cancer Center (HICCC) at Columbia University, the Roswell Park Comprehensive Cancer Center (RPCCC), the University of British Columbia and Omniseq, a commercial biotech spin-off that is majority owned by RPCCC. In Aim 1 (UH2 phase), we perform the analytical validation of the assay including validation of gene reference controls, RNA extraction quality, and reporter binding density. The milestone for moving to the UH3 phase (Aim 2) will be submission to the NYS Clinical Laboratory Evaluation Program. In the UH3 clinical validation phase, we first evaluate two additional retrospective sample populations (from HICCC and RPCCC). As part of this study we will also correlate the 53-gene signature with state of the art immune indices including quantitative multiplex immunofluorescence (qmIF) to assess CD8 to CD68 ratio, a metric recently defined (by our group) to correlate with survival. We then perform the definitive PRA analysis using the E1697 samples.